The long-term objective of the proposed research is to advance our understanding in two areas: 1) The structure, function, and regulation of mammalian cell surface receptors. 2) The molecular basis of eukaryotic gene expression and regulation. These problems will be addressed in two separate but related systems, each responsible for providing mammalian cells with cholesterol. The model for receptor studies is the LDL (low density lipoprotein) receptor which provides the cell with cholesterol from the extracellular environment (project 1). Coordination of receptor activity with intracellular metabolism will be studied focusing on the complex regulation of mammalian HMG-CoA reductase, the rate-limiting enzyme in de novo biosynthesis of cholesterol from acetate (project 2). Project 1. Our experimental approach employs recombinant DNA technology, nucleic acid biochemistry, and molecular genetics. Initial experiments outline the isolation and characterization of the gene(s) encoding the LDL receptor. Subsequent studies will be aimed at (1) identification of functionally important features of the receptor and its gene, (2) studies of the molecular basis of differential receptor expression, and (3) generation of new phenotypically receptor-negative cell lines by mutagenesis in order to identify additional genes whose products are required for receptor-mediated endocytosis or receptor regulation. Project 2. The experimental approach begins with isolation of the gene(s) encoding HMG-CoA reductase. These cloned DNA sequences will be used to study the role of transcriptional and post-transcriptional events in reductase induction and suppression. Mutants possessing altered reductase activity or regulation will be generated in somatic cells, and characterized biochemically and genetically. The proposed research examines directly the genetic and moleculr basis of Familial Hypercholesterolemia Type II, a major human health problem whose principle clinical manifestations are atherosclerosis and coronary heart disease.